Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.

Rezidivierte und refraktäre Hodentumoren waren bis zu Beginn der 80er Jahre nur selten kurativ behandelbar. Mit Einführung der Hochdosischemotherapie in Verbindung mit autologer Stammzellreinfusion, konnte eine kurative Behandlungsoption auch in dieser prognostisch ungünstigen Situation in der Klinik etabliert werden. Die vorliegende Arbeit beschreibt die Ergebnisse der ersten Phase I/II Studie zur klinischen Etablierung dieses Therapieverfahrens ebenso wie verschiedene nachfolgende Untersuchungen zur Optimierung der Hochdosischemotherapie. Eine "matched-pair" Analyse konnte zumindest im retrospektiven Vergleich, den Nutzen dieses neuen Therapieverfahrens im Vergleich zu einer konventionell-dosierten Behandlung belegen. / Until the beginning of the 1980ies relapsed and refractory germ-cell tumors were rarely cured. With the introduction of high-dose chemotherapy in combination with autologous stem cell reinfusion, a curative treatment option could be established in this prognostically unfavorable situation. The present work describes the results from the initial phase I/II studies that established this new treatment as well as the results of several subsequent trials to optimize this new procedure. Finally, the results of a "matched-pair" analysis is presented that demonstrates the superiority of this new treatment as compared to conventional-dose chemotherapy.

La stérilisation des espèces aquacoles est recherchée en aquaculture pour remédier aux problèmes de reproduction intempestive et risques de pollution génétique de la reproduction des poissons d'élevage échappés avec des espèces endémiques. Nous avons cherché à caractériser certains des mécanismes mis en jeu lors d'une stérilisation induite par de fortes températures chez une espèce de poissons thermosensible d'intérêt majeur en aquaculture, le tilapia du Nil, Oreochromis niloticus. L'effet d'une température élevée de 37°C sur le développement gonadique a été étudié pendant la différenciation sexuelle chez des alevins et pendant la maturation sexuelle chez des juvéniles, provenant de descendances mixtes (XXXY) et monosexes femelles (XX) /mâles (XY). L'analyse immunohistochimique de la protéine vasa montre que des traitements à 37°C provoquent une diminution du nombre de cellules germinales (CG) qui résulte d'une augmentation du taux d'apoptose et/ou d'une réduction du taux de prolifération de ces cellules, aboutissant à une stérilité partielle et transitoire ou complète et permanente. L'expression du gène vasa, marqueur des CG, est inhibée dans les gonades des poissons traités à 37°C pendant un minimum de 60 jours ; ceci est corrélé avec la réduction du nombre de CG dans ces gonades. La baisse du niveau d'expression des gènes cyp19a1a et amh, respectivement marqueurs de cellules somatiques femelles et mâles, suggère que la température de 37°C affecte également le nombre ou la fonctionnalité des cellules de la granulosa chez les femelles et de Sertoli chez les mâles. Un traitement de 60 jours est nécessaire pour induire de tels effets et semble impacter préférentiellement les gonades femelles. Ce travail confirme que les fortes températures induisent une réduction du nombre de CGs, en modifiant la balance entre les taux d'apoptose et de prolifération cellulaire, conduisant à des stérilités partielles ou totales. / The sterilization of farmed fishes is searched in aquaculture to remedy in the problems of inconvenient reproduction and risk of genetic pollution of reproduction of escaped farmed fishes into the natural environment with endemic species. We characterized some of the mechanisms involved during induced-sterilization by high temperatures in the Nile tilapia, Oreochromis niloticus, a thermosensitive species of major interest in fish farming. The effect of 37°C-elevated temperature on gonadal development was studied during sexual differentiation in fry and during sexual maturation in juvenile, from mix-sexed (XX/XY), all-genetic female (XX) and male (XY) progenies. The immunochemistry analysis of vasa protein shows that 37°C-treatment causes germ cell (CGs) decrease, resulting from an increase of apoptosis rate and/or reduction of cell proliferation, leading to partial and transitory or complete and permanent sterilization.This work confirms that high temperatures induce a decrease of germ cell number, modifying the balance between rates of cell apoptosis and cell proliferation, leading to partial or complete sterilization.The expression profile of vasa gene, marker of germ cells, is inhibited in gonads of fish treated at least during 60 days at 37°C ; that is correlated with the reduction of germ cell number. The reduction of expression levels of cyp19a1a and amh genes, respectively markers of female and male somatic cells, suggests that the 37°C-temperature also affects the number or function of granulosa cells in females and Sertoli cells in males. A treatment of 60 days is necessary to induce such effects and seems to impact preferentially female gonads.

Fxrα est le récepteur nucléaire des acides biliaires, exprimé majoritairement dans le foie, l'intestin, les reins et les glandes surrénales. L'intérêt pour ce dernier est devenu croissant au cours des dernières années, de part le rôle central qu'il joue dans le contrôle de l'homéostasie du cholestérol, des acides biliaires, des triglycérides ou encore du glucose. Plus récemment, Fxrα ainsi que ses ligands, les acides biliaires, ont été localisés dans le testicule, soulevant la question du rôle potentiel de Fxrα dans cet organe, et plus généralement dans la fonction de reproduction mâle. Mais les études menées à ce sujet restent jusqu'à présent peu nombreuses, et focalisées sur son implication dans le contrôle du métabolisme des stéroïdes : l'activation in vivo de Fxrα par un agoniste synthétique conduit ainsi chez l'adulte à court terme à une répression de la stéroïdogenèse. Outre son rôle dans le contrôle de l'activité endocrine des cellules de Leydig, l'impact de l'activation in vivo de Fxrα sur la physiologie plus globale du testicule n'a jamais été abordé à ce jour. De telles études seraient pourtant pertinentes étant donné que Fxrα est ciblé pour le traitement de pathologies métaboliques telles que la dyslipidémie ou le diabète. Dans ce contexte, l'objectif de ce travail de thèse était d'étudier le rôle de Fxrα dans la physiologie et la pathophysiologie du testicule, en s'appuyant sur l'analyse d'un modèle murin dont le gène codant Fxrα a été invalidé. Nos résultats démontrent que : 1) la perte de Fxrα prédispose le testicule à une sur-mortalité des cellules germinales dans un contexte pathologique de cholestase ; 2) la sur-activation de la signalisation Fxrα au cours de la puberté conduit à un défaut de la différenciation germinale, associée à une altération de la fonction endocrine du testicule ; 3) outre la régulation de la stéroïdogenèse dans les cellules de Leydig, Fxrα participe au contrôle des fonctions sertoliennes et de la prolifération et / ou différenciation des cellules germinales souches. L'ensemble de ces données définissent Fxrα comme un nouvel acteur impliqué dans le contrôle de la physiologie testiculaire et devraient être prises en considération quant-à l'utilisation de molécules agonistes et / ou antagonistes de Fxrα dans le cadre du traitement de pathologies métaboliques. / Fxrα is the bile acid nuclear receptor, predominantly expressed in liver, intestine, kidney and adrenal glands. In recent years, interest in Fxrα has been increasing due to its central role in the control of cholesterol, bile acids, triglycerides or glucose homeostasis. More recently, Fxrα and its ligands, bile acids, have been detected in the testis pointing out its potential involvement in this tissue and more widely in the male reproductive functions. However, the few studies on this topic focused essentially on Fxrα involvement in the control of steroids metabolism. Indeed, activation of Fxrα in vivo with a synthetic agonist leads to short-term steroidogenesis repression in the adult. In vivo the impact of alteration of Fxrα signaling on the global testis physiology has never been explored so far. Such studies would be pertinent considering that Fxrα is a target for the treatment of metabolic diseases such as dyslipidemia or diabetes. In this context, the aim of my work was to study the implication of Fxrα in testis physiology and physiopathology by analyzing a knock out mouse model for Fxrα. Our results show that: 1) the loss of Fxrα increase germ cell mortality in the testis in a disease context of cholestasis ; 2) over-activation of Fxrα signaling during puberty leads to germ cell differentiation defects, associated with an alteration of testis endocrine function ; 3) besides steroidogenesis control in Leydig cell, Fxrα is involved in Sertoli cell functions and spermatogonial stem cell proliferation and/or differentiation. Taken together, these data define Fxrα as a new actor involved in the control of testis physiology, and should be taken into consideration regarding the use of Fxrα agonistic or antagonistic ligands for the treatment of metabolic diseases.

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Testicular germ cell tumors (TGCTs) are thought to arise during early embryogenesis due to the arrest of germ cell differentiation at primordial germ cells (PGCs) or gonocytes. Oxidative stress (OS) is implicated in cancer development as a factor leading to DNA damage. Reactive oxygen species (ROS) -induced instability occurs as a series of progressive steps. The cell has several defense mechanisms against the deleterious effect of ROS (e.g. antioxidants and DNA repair). When the defense mechanisms are exhausted by increasing OS, DNA damage leads to genomic instability with subsequent mutations that can be transmitted during cell division. On the other hand, male infertility is a representation of testicular dysgenesis syndrome, which carries a risk for TGCTs development. The mechanisms underlying both TGCTs and male infertility are thought to be overlapping to some extent. The central hypothesis of this work is that OS induces germ line genomic instability leading to testicular germ cell tumors. To test this hypothesis, mouse germ cell lines were established and subjected to different doses of OS in the form of H2O2. The mutation frequency was associated with the treatment dose 2 uM at days 3, 6, and 9 (p<0.001, p<0.001, and p<0.0003, respectively). The mBAT27 marker showed a mutation frequency fitting quadratic response surface regression. The mutation frequencies pointed to the possible role of OS leading to accumulation of DNA damage and initiating events that lead to TGCTs development that may occur early in life, possibly during the prenatal period. In addition, different panels of microsatellite markers from across the genome were analyzed to test for differential instability in both somatic cells and germline cells. Blood and semen samples from 18 infertile patients and 7 ethnically matched controls were used. Microsatellite markers were selected; 26 on the Y chromosome, 16 on the X chromosome, and 20 on different autosomes. Microsatellite instability was detected in markers located near genes responsible for testis development, spermatogenesis, cell differentiation, and proteins involved in mismatch repair mechanisms. This supports the hypothesis that testicular germ cell tumors may arise during early embryogenesis through acquiring multiple mutations that accumulate over time.

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Endogenous retrovirus ERVWE1 is an integral part of the human genome. In the course of evolution, a protein encoded by the env gene of this retrovirus - Syncytin-1 - has gained unique function in human development. It mediates cell-to-cell fusion of placental cytotrophoblasts. Receptor that binds to Syncytin-1 is expressed in different cell types. Syncytin-1-mediated fusion is essential in placenta, but it can cause disruption of tissue integrity in other cell types. ERVWE1 expression is regulated by promoter DNA methylation, transcription factor GCM1 and efficient mRNA splicing. This thesis concerns the ERVWE1 expression and its regulation in non-placental tissues. It was found out that the moderate GCM1 overexpression was not sufficient to induce Syncytin-1 expression. Neither treatment with DNA demethylation agent 5-azacytidine nor with Syncytin-1 activator forskolin was able to manage Syncytin-1 expression. This thesis extends previous findings concerning high syncytin-1 expression in seminomas. In same tissues, there was found elevated TET1 expression on mRNA level in comparison with controls. The presence of the TET1 demethylation enzyme can influence ERVWE1 promoter DNA methylation. Previously unreported splicing variant of TET1 has been found during the construction of human TET1 expression...